SV40 T/t Interactions with Pocket Proteins

我们已经积累的证据表明，在S期，Rb蛋白的非(较)磷酸化形式具有独特的作用，它被SV40小T抗原阻断。具体地说，Rb可以抑制DNA损伤后DNA片段的重新复制，至少部分是通过定位并与多个基因组复制起始元件形成复合体来实现的。为了准备损伤后复制位点的定位，RB也必须定位在染色质部分，我们已经证明这个过程是PP2A依赖的。因此，小的t阻止了Rb/染色质的定位，从而阻止了它在DNA损伤后定位到复制起始点的能力。在这方面，小t作用的一个最终产物是细胞DNA的内源性复制。此外，我们还发现Nijmegen Break综合征基因的产物Nbs1是一种迄今未被识别的大标签结合蛋白；Nbs1像Rb一样参与细胞复制子和病毒基因组的再复制抑制；当Nbs1结合时，标签可以覆盖这一活性。后一种效应可能有助于T诱导基因组再复制以及对包含SV40复制起点的环状DNA结构的完全自主复制。在这一应用中，我们建议寻找：1)生化证据，揭示RB和NBS1如何抑制基因组再复制；2)洞察PP2A RB/染色质进入过程在细胞周期中的哪里适用；3)关于PP2A如何在这一过程中进行生化操作的信息；以及4)证据表明RB和/或其他口袋蛋白是否在其肿瘤共转化功能的执行过程中作为关键的小t靶标。

We have accumulated evidence pointing to a distinctive role of the un(der)phosphorylated form of the Rb protein during S phase that is blocked by SV40 small t Ag. Specifically, Rb can suppress the rereplication of DNA segments after DNA damage, at least in part by homing to and forming complexes with multiple genomic replication initiation elements. In preparation for post-damage replication site localization, Rb must also localize in the chromatin fraction, a process that we have shown is pp2A-dependent. Thus, small t blocks Rb/chromatin localization and, thereby, its ability to home to replication initiation sites after DNA damage. One end product of small t action in this regard was endoreduplicafion of cellular DNA. In addition, we have found that the product of the Nijmegen Break Syndrome gene, Nbs1, is a heretofore unappreciated large TAg binding protein; that Nbs1, like Rb, participates in rereplication suppression-both of cellular replicons and the viral genome; and that TAg can, upon Nbs1 binding, override this activity. The latter effect may well contribute to the established ability of T in inducing genomic rereplication as well as fully autonomous replication of circular DNA structures that contain an SV40 replication origin. In this application, we propose to search for: 1) biochemical evidence that sheds light on how Rb and Nbs1 suppress genomic rereplication; 2) for insights into where during the cell cycle the pp2A Rb/chromatin entry process is applicable; 3) for information on how pp2A operates biochemically in this process, and 4) for evidence indicating whether or not Rb and/or other pocket proteins serve as key small t targets during the performance of its neoplastic co-transforming function in human cells.